|Yokomi, Raymond - Ray|
Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2013
Publication Date: 8/8/2013
Citation: Loconsole, G., Onelge, N., Yokomi, R.K., Abou-Kubaa, R., Savino, V., Saponari, M. 2013. Rapid differentiation of citrus Hop stunt viroid variants by use of real-time RT-PCR and high resolution melting analysis. Molecular and Cellular Probes. 27:221-229. Interpretive Summary: Viroids are small RNA molecules that cause various symptoms in citrus. Some viroids are beneficial and can be used to reduce tree size for ease of harvesting and close row spacing while others cause debilitating disease. The hop stunt viroid contains pathogenic (cachexia) and non-pathogenic strains affecting citrus. Rapid detection and differentiation of hop stunt viroid strains are needed in citrus budwood certification and clean stock programs to replace biological indexing which requires 6-12 months. A polymerase chain reaction PCR assay was developed using unique sequences in the hop stunt viroid genome associated with the ability to cause disease. The assay distinguished citrus hop stunt viroids into groups associated with cachexia versus non-cachexia variants. This assay was used to accurately identify and distinguish 50 known hop stunt viroid-infected samples from field sources in California, Italy, Spain, Syria and Turkey. This diagnostic method will facilitate maintaining viroid-free budwood in citrus nurseries, verify viroid infection and identify infected trees for removal.
Technical Abstract: The RNA genome of Hop stunt viroid (HSVd) contains five to six nucleotides in a variable (V) domain, called the cachexia expression motif, which is associated with pathogenic and non-pathogenic variants in citrus. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson’s Special mandarin followed by direct nucleotide sequence analysis from infected plants. For these reasons, real-time RT-PCR assays were improved that allowed simultaneous detection and differentiation of cachexia versus non-cachexia HSVd variants. Primers that targeted several single nucleotide polymorphisms in the cachexia expression motif were developed which produced real-time RT-PCR amplicons that separated variants by High-Resolution Melting Temperature (HRM) analysis. Two separate assays were developed based on different dye chemistries: (i) EVA Green (ii) TaqMan. In both cases, HSVd variants were separated into three clusters by distinct melting temperatures with confidence level greater than 98%. Specificity of the HRM assays was confirmed by nucleotide sequencing of representative samples within each cluster and validated by testing 50 known HSVd-infected samples from sources in California, Italy, Spain, Syria and Turkey. This is the first report of a sensitive protocol to detect and differentiate HSVd variants associated with different biological characters. Although HSVd also is found in crops other than citrus, cachexia-variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid detection of HSVd variants, thus, is important for management of cachexia and reduces the need for biological indexing and/or sequence analysis.